Multifunctional micellar nanoparticle-based drug and targeting agent system

ABSTRACT

Embodiments provide systems, methods, and compositions for nanoparticle-based drug delivery to target cells or tissues. A drug delivery system may include a nanoparticle with a targeting component and a therapeutic component. The nanoparticle may have a predetermined number or valence of targeting molecules for multivalent interaction with a target cell or tissue. Binding of the targeting molecules to the target cell may result in receptor-mediated uptake of the nanoparticle by the target cell. The therapeutic component may be subsequently released within an endocytic vesicle of the target cell. Nanoparticle-based drug delivery systems as described herein may provide improved efficacy and/or reduced toxicity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/356,121, filed May 2, 2014, issued as U.S. Pat. No. 9,872,870, whichis a National Stage filing under 35 U.S.C. § 371 of InternationalApplication No. PCT/US2012/063614, filed Nov. 5, 2012, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Patent ApplicationNo. 61/628,741, filed Nov. 4, 2011, which applications are incorporatedherein by reference.

SEQUENCE LISTING

The present application includes a Sequence Listing in electronic formatas a text file titled “Seq_Listing.txt” which was created on Nov. 5,2012 and has a size of 2,241 bytes. The contents of txt file“Seq_Listing.txt” are incorporated by reference herein.

BACKGROUND

Multiple myeloma (MM) is a B-cell malignancy characterized byproliferation of monoclonal plasma cells in the bone marrow (BM).Despite recent advances in treatment strategies and the emergence ofnovel therapies, it remains incurable (median survival of 4-5 years) dueto the development of drug resistance. A major factor that leads to drugresistance in MM patients is the adhesion of MM cells to the BM stroma.This results in cell-adhesion-mediated drug resistance (CAM-DR), whichrenders the MM cells in the BM microenvironment less sensitive tochemotherapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments will be readily understood by the following detaileddescription in conjunction with the accompanying drawings. Embodimentsare illustrated by way of example and not by way of limitation in thefigures of the accompanying drawings.

FIG. 1 illustrates an embodiment of a drug delivery nanoparticle;

FIGS. 2A-2C illustrate components of the drug delivery nanoparticle ofFIG. 1;

FIGS. 3A-3C are schematic diagrams of drug delivery nanoparticle uptakeand drug release in a target cell;

FIG. 4 illustrates the structure of VLA-4 antagonist peptide(VLA-4-pep);

FIG. 5 illustrate expression of VLA-4 subunits α4- and β1-integrins inMM.1S, NCl-H929, U266, and IM9 cell lines, as determined by flowcytometry;

FIG. 6 illustrate binding of VLA-4-pep to U266, NCl-H929, and 1M-9 celllines with apparent K_(d) of ˜250 nM (FIG. 6a ) and inhibition ofadhesion of MM cell lines to fibronectin-coated plates by VLA-4-pep(FIG. 6b );

FIG. 7 illustrate synthesis and characterization of VLA-4 targeting, Doxconjugated multifunctional nanoparticles (NPDox/VLA-pp);

FIG. 8 illustrate cellular uptake studies with drug deliverynanoparticles;

FIG. 9 illustrates induction of cytotoxicity in MM cells byNPDox/VLA-4-pep;

FIG. 10 illustrate induction of DNA DSBs and apoptosis in MM cells byNPDox/VLA-pep;

FIG. 11 illustrate inhibition of adhesion of MM cells to fibronectin andovercoming of CAM-DR by NPDox/VLA-4-pep;

FIG. 12 illustrate in vivo characterization of NPDox/VLA-4-pep in asubcutaneous xenograft model of MM;

FIG. 13 illustrates a flow diagram of a method for constructing a drugdelivery nanoparticle; and

FIG. 14 illustrates a flow diagram of a method for designing and using adrug delivery nanoparticle to treat a target cell or tissue, all inaccordance with various embodiments.

DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS

In the following detailed description, reference is made to theaccompanying drawings which form a part hereof, and in which are shownby way of illustration embodiments that may be practiced. It is to beunderstood that other embodiments may be utilized and structural orlogical changes may be made without departing from the scope. Therefore,the following detailed description is not to be taken in a limitingsense, and the scope of embodiments is defined by the appended claimsand their equivalents.

Various operations may be described as multiple discrete operations inturn, in a manner that may be helpful in understanding embodiments;however, the order of description should not be construed to imply thatthese operations are order dependent.

The description may use perspective-based descriptions such as up/down,back/front, and top/bottom. Such descriptions are merely used tofacilitate the discussion and are not intended to restrict theapplication of disclosed embodiments.

The terms “coupled” and “connected,” along with their derivatives, maybe used. It should be understood that these terms are not intended assynonyms for each other. Rather, in particular embodiments, “connected”may be used to indicate that two or more elements are in direct physicalor electrical contact with each other. “Coupled” may mean that two ormore elements are in direct physical or electrical contact. However,“coupled” may also mean that two or more elements are not in directcontact with each other, but yet still cooperate or interact with eachother.

For the purposes of the description, a phrase in the form “A/B” or inthe form “A and/or B” means (A), (B), or (A and B). For the purposes ofthe description, a phrase in the form “at least one of A, B, and C”means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).For the purposes of the description, a phrase in the form “(A)B” means(B) or (AB) that is, A is an optional element.

The description may use the terms “embodiment” or “embodiments,” whichmay each refer to one or more of the same or different embodiments.Furthermore, the terms “comprising,” “including,” “having,” and thelike, as used with respect to embodiments, are synonymous.

In various embodiments, methods, apparatuses, and systems for targeteddrug delivery are provided. In exemplary embodiments, a computing systemmay be employed to perform, or to control devices employed to perform,one or more methods as disclosed herein.

Embodiments described herein provide nanoparticle-based drug deliverysystems and methods. In some embodiments, a drug delivery system mayinclude a nanoparticle with a targeting component and a therapeuticcomponent. The nanoparticle may have a predetermined number or valenceof targeting molecules for multivalent interaction with a target cell ortissue. Binding of the targeting molecules to the target cell may resultin receptor-mediated uptake of the nanoparticle by the target cell. Thetherapeutic component may be subsequently released within an endocyticvesicle of the target cell.

As used herein, a “targeting molecule” or “targeting agent” is apeptide, cyclic peptide, peptidomimetic, or other molecule that binds toa targeted molecule (e.g., a cell surface molecule of a cell targetedfor treatment, and/or an extracellular matrix component). Optionally,the binding affinity of the targeting molecule may be in the range of 1nM to 1 μM. In some embodiments, the targeting molecule may be anantagonist of a receptor on the surface of a targeted cell.

As used herein, a “therapeutic agent” can be any type of molecule usedin the treatment, cure, prevention, or diagnosis of a disease or othermedical condition. Examples of therapeutic agents include, but are notlimited to, drugs (e.g., anti-cancer drugs, antibiotics) and nucleicacids (e.g., siRNA, DNA). Specific examples of therapeutic agentsinclude, but are not limited to, bortezomib, carfilzomib, andplatinum-containing drugs (e.g., cisplatin, carboplatin, derivativesthereof).

FIG. 1 illustrates an embodiment of a drug delivery nanoparticle 100, inaccordance with various embodiments. A nanoparticle 100 may be designedfor use to prevent, treat, or cure a disease or other medical condition.As illustrated, nanoparticle 100 may have a hydrophobic interior portion120 surrounded by an outer portion that includes polar head groups 110,water-soluble polymers 130, targeting molecules 140, and therapeuticagent molecules 150. Nanoparticle 100 may include at least three groupsof component molecules, which are shown in FIG. 1b as components 102,104, and 106. As used herein, the term “component” may refer to onemolecule of a particular species (e.g., one molecule of component 102)or to a plurality of molecules of a particular species (e.g., two ormore molecules of component 102).

Nanoparticle 100 may include components 102, 104, and/or 106 in apredetermined ratio. As used herein, a “molecular ratio” may be providedto indicate the number of molecules of two or more components (e.g.,102, 104, 106) in a nanoparticle. The number of molecules of a componentin a nanoparticle may also be described in terms of a “mole percentage,”which is calculated by dividing the number of molecules of thatcomponent by the number of molecules in the nanoparticle. For example,in a nanoparticle with about 90 component molecules, 60 of which arecomponent 102, 20 of which are component 114, and 10 of which arecomponent 116, the “molecular ratio” of the components (112:114:116) is60:20:10, and the mole percentages of the three components are 67%, 22%,and 11%, respectively.

FIGS. 2a-2c are schematic diagrams of components 102 (FIG. 2a ), 104(FIG. 2b ), and 106 (FIG. 2c ). Components 102, 104, and 106 may eachinclude a lipid molecule (122, 124, and 126, respectively) coupleddirectly or indirectly to one or more additional molecules. Typically,lipid molecules 122, 124, and 126 are amphipathic lipid molecules, eachwith a polar/hydrophilic portion 110 and a non-polar/hydrophobic portion108. Optionally, some or all of the lipid molecules may bephospholipids. In some examples, lipid molecules 122, 124, and/or 126may be different chemical species. Alternatively, two or more of lipidmolecules 122, 124, and 126 may be the same chemical species. Forexample, lipid molecule 122 and lipid molecule 124 may be the samechemical species (e.g., DSPE) and lipid molecule 126 may be a differentchemical species (e.g., DPPE-GA).

Referring now to FIG. 2a , component 102 may be configured to enhancethe stability and/or circulation time of nanoparticle 100. In someembodiments, component 102 may include a polymer 112 conjugated orotherwise coupled to lipid molecule 122. Polymer 112 may be awater-soluble polymer, such as polyethylene glycol (PEG). In otherembodiments, polymers 112 may be polymeric sugars, PLGA, and/or otherbiocompatible water soluble molecules. In one embodiment, component 102may be a lipid-PEG block copolymer (e.g., DSPE-PEG2000).

As shown in FIG. 2b , component 104 may be configured to targetnanoparticle 100 to a particular cell or tissue type. In someembodiments, component 104 may include a targeting molecule 140 and apolymer 112 conjugated or otherwise coupled to lipid molecule 124. Forexample, targeting molecule 140 may be coupled to a first end of polymer112, and lipid molecule 124 may be coupled to an opposite second end ofpolymer 112.

Targeting molecule 140 may be, or may include, a peptide, a cyclicpeptide, a peptidomimetic, and/or a small molecule. Targeting molecule140 may be configured to bind to a component of extracellular matrix(ECM) and/or to a cell surface molecule of a target cell. Examples ofsuch cell surface molecules include, but are not limited to, integrins,cadherins, selectins, and syndecans. In some embodiments, the cellsurface molecule of the target cell may be a receptor, and targetingmolecule 140 may be a ligand for the receptor. In some embodiments,targeting molecule 140 may be an antagonist of the receptor. Forexample, targeting molecule 140 may be an antagonist that interfereswith a function of the target cell (e.g., adhesion) upon binding to thereceptor. Alternatively, targeting molecule 140 may be an agonist orpartial agonist of the receptor.

In some examples, the cell surface molecule of the target cell/tissuemay be a molecule that is expressed only by the target cells/tissue(s).In other examples, the cell surface molecule of the target cell/tissuemay be a molecule that is expressed by both target cells and non-targetcells, but is present in fewer numbers and/or in a different spatialarrangement on non-target cells than on target cells.

In some embodiments, binding of the targeting molecule 140 to the cellsurface molecule of the target cell may trigger endocytosis ofnanoparticle 100 by the target cell. Optionally, the cell surfacemolecule may play a role in the adhesion of the target cell to ECMand/or the surrounding environment, and binding of the targetingmolecule 140 to the target cell may reduce or disrupt this adhesion.

Targeting molecule 140 may be an antagonist of a target cell surfacereceptor. In some embodiments, targeting molecule 140 may be a VLA-4antagonist. In the specific example described further below, targetingmolecule 140 is a VLA-4 antagonist peptide (“VLA-4-pep”, YCDPC; SEQ IDNO: 1) that is configured to bind to fibronectin and to VLA-4. Thestructure of VLA-4-pep is shown in FIG. 3. Other examples of VLA-4antagonists that could be used as targeting molecules include, but arenot limited to, the peptide sequence CFLDFP (SEQ ID NO: 2), peptidesequences with a consensus LDV sequence, cyclic peptides with an RCDmotif, sequences based on (X)CDPC (SEQ ID NO: 3), XC(Z)PC (SEQ ID NO:4), XCA(Z)C (SEQ ID NO: 5), (X)CSPC (SEQ ID NO: 6), YC(X)C (SEQ ID NO:7), or RC(X)PC (SEQ ID NO: 8) core structures (where X and Z arevariable amino acids), peptides derived from fibronectin CS-1, peptidesderived from fibronectin RGD tripeptide, peptides derived fromfibronectin RGD and vascular cell adhesion molecule-1, peptides derivedfrom anti-α₄ monoclonal antibody, and other VLA-4 antagonists known inthe art. Additional examples of VLA-4 antagonists are described in thefollowing references: Jackson, David Y. et al., “Potent a4b1 PeptideAntagonists as Potential Anti-Inflammatory Agents,” J. Med. Chem, vol.40, pp. 3359-3368, 1997; Lin, Ko-Chung and Castro, Alfredo C., “Verylate antigen 4 (VLA4) antagonists as anti-inflammatory agents,” CurrentOpinion in Chemical Biology, vol. 2, pp. 453-457, 1998; and Tilley,Jefferson W., “Very late antigen-4 integrin antagonists,” Expert Opin.Ther. Patents, vol. 18, no. 8, pp. 841-859, 2008, all of which arehereby incorporated by reference herein

Alternatively, targeting molecules 140 may be antagonists and/or ligandsof other receptors. Examples of other targeting molecules include, butare not limited to, folate (binds folate receptor), RGD peptidesequences against the αvβ3 integrin, and peptide antagonists of theHuman Epidermal Growth Factor Receptor 2 (HER2). In some embodiments,the binding affinity of targeting molecule 140 to the target cellsurface receptor is in the range of 1 nM to 1 μM. Binding affinities inthis range may allow for binding enhancements due to multivalentinteractions.

Referring now to FIG. 2c , component 106 may include a therapeutic agent150 coupled to lipid molecule 126. Optionally, therapeutic agent 150 maybe coupled to lipid molecule 126 by a pH-sensitive bond 118. In someembodiments, pH-sensitive bond 118 may be acid-labile. For example,pH-sensitive bond 118 may be a hydrazone (HN-NH) bond.

Therapeutic agent 150 can be or can include any type of drug for whichdelivery to a target cell or tissue is desired. In one example,therapeutic agent 150 is doxorubicin. However, in other examplestherapeutic agent 150 may be another drug, a nucleic acid (e.g., siRNA,DNA, etc.), or some combination thereof. In some embodiments,therapeutic agent 150 may be an antibiotic or an anti-cancer drug.Antibiotics and anti-cancer drugs are known in the art. Examples ofsuitable therapeutic agents 150 may include, but are not limited to,bortezomib, carfilzomib, and platinum-containing drugs (e.g., cisplatin,carboplatin, or a derivative thereof). Optionally, a nanoparticle mayinclude two or more different therapeutic agents 150 coupled to the samelipid molecule or to different lipid molecules.

Nanoparticles 100 may be constructed to include components 112, 114, and116 in predetermined molecular ratios. Stated in another way,nanoparticles 100 may be constructed using methods described herein toresult in predetermined molar percentages of components 112, 114, and116 in most or all of the nanoparticles. The molar/molecular ratios ofcomponents 102, 104, and 106 in nanoparticle 100 may vary amongembodiments. In some examples, the molar percentage of component 102 maybe 60-100% (e.g., 60-70%, 60-90%, 70-90%, 75-95%, 80-90%, 80-95%, or90-100%). In other examples, the molar percentage of component 104 maybe 0-60% (e.g., 0-5%, 0-40%, 1-40%, 10-30%, 15-35%, 20-30%, 20-40%, or30-40%). In still other examples, the molar percentage of component 106may be 0-25% (e.g., 0-5%, 1-10%, 0-20% 1-20%, 5-15%, 5-20%, or 10-20%).In a specific embodiment, component 102 may be a pegylated phospholipid(e.g., DSPE-PEG2000), and may be present in nanoparticle 100 at a molarpercentage of 60% or greater in order to maintain a low CMC. Optionally,the molar percentage of component 112 may be greater than the molarpercentage of component 114, which may be greater than the molarpercentage of component 116. Alternatively, the molar percentage ofcomponent 116 may be greater than the molar percentage of component 114.

In a particular embodiment, component 112 is a pegylated phospholipid(e.g., DSPE-PEG2000), component 114 is a peptide-PEG-phospholipidconjugate (e.g., VLA-4-pep/DSPE-PEG2000), and component 116 is ananti-cancer drug conjugated to a phospholipid (e.g., Dox/DPPE-GA). Inthis embodiment, nanoparticles may include 80-100 component molecules,or about 90 component molecules. The molar percentages of components112, 114, and 116 may be about 67%, about 22%, and about 11%,respectively. For example, nanoparticles may have about 90 componentmolecules, with a predetermined ratio of component 102:component104:component 106 of about 60:20:10.

FIGS. 3a-3c are schematic diagrams of drug delivery nanoparticle uptakeand drug release in a target cell, in accordance with variousembodiments. FIG. 3a shows nanoparticle 100 binding to a receptor 170 ona target cell 103 via targeting molecule 140. While only a singlebinding event is illustrated, it is to be understood that multipletargeting molecules 140 may bind to corresponding receptors 170 on thetarget cell. As illustrated in FIG. 3b , the binding of targetingmolecule(s) 140 to receptor(s) 170 may cause receptor-mediated uptake ofnanoparticle 100 by target cell 103. Thus, nanoparticle 100 may beinternalized within a vesicle 190 (e.g., an endosome/lysosome).Nanoparticle 100 may release therapeutic agent 150 within vesicle 190.The release of therapeutic agent 150 may be triggered, for example, by areduction in pH within vesicle 190. In some embodiments, therapeuticagent 150 may be coupled to nanoparticle 100 by a pH-sensitive oracid-labile bond that is disrupted in acidic conditions (e.g., at ˜pH5.5).

Thus, a nanoparticle may be designed with targeting molecules toselectively target cells expressing a surface molecule and to deliver atherapeutic agent to the target cells. In some embodiments, thetargeting molecule may bind to a surface molecule required for cellularadhesion. Nanoparticles configured to engage in multiple low affinityinteractions with a target cell may better distinguish one cell typefrom another, resulting in improved selectivity. Therefore, thetargeting molecules of a nanoparticle may be peptides selected for abroad range of binding affinities. Methods described herein may be usedto construct nanoparticles with a desired ratio of targeting moleculesto other components. Such methods allow the production of nanoparticleswith a desired valence of the targeting molecules, and minimalbatch-to-batch variation. Constructing nanoparticles with tightlycontrolled numbers/valences of targeting molecules may allow foroptimization of multivalent interactions with the target cell forimproved binding affinity and specificity of ligand-receptorinteractions.

In some embodiments, a multifunctional nanoparticle may include atargeting component configured to bind to target surface receptors of amultiple myeloma (MM) cell and a therapeutic component configured toinhibit the survival of the MM cell. The binding of the targetingcomponent to the target surface receptors may reduce or overcome celladhesion mediated drug resistance (CAM-DR) in the MM cell, therebypreserving or enhancing the efficacy of the therapeutic component.Specific examples of this embodiment are described in further detailbelow.

VLA-4-mediated adhesion of multiple myeloma (MM) cells to the bonemarrow stroma confers MM cells with cell-adhesion-mediateddrug-resistance (CAM-DR). In the examples described below, micellarnanoparticles were used as dynamic self-assembling scaffolds to presentVLA-4-antagonist peptides and doxorubicin conjugates simultaneously toselectively target MM cells and to overcome CAM-DR. Very late antigen-4(VLA-4; also known as α4 β1 integrin) is a cell surface heterodimerexpressed on cancers of hematopoietic origin such as lymphomas,leukemias, and MM. In MM, VLA-4 is a key adhesion molecule that acts asa receptor for the extracellular matrix protein fibronectin and thecellular counter-receptor VCAM-1 (expressed on the BM stromal cells).VLA-4 plays a critical role in CAM-DR of MM cells and providesresistance to first line chemotherapeutics such as doxorubicin (Dox).Importantly, inhibition of MM cell adhesion to the BM microenvironmentvia α4-integrin blocking antibodies or α4-siRNA overcomes drugresistance in MM cells.

Enhanced accumulation of nanoparticles may be observed in tumor tissuedue to the leaky vasculature found in the angiogenic vessels seenpredominantly in solid tumors. Recent evidence in research hasestablished that angiogenesis also plays a major role in somehematologic malignancies including MM. In line with these findings,liposomal doxorubicin (Doxil) has shown beneficial clinical outcome inMM patients when used in combination with vincristine and dexamethasone(VAD therapy), and has recently been FDA approved in combination withbortezomib in the treatment of relapsed or refractory MM. Despite theestablishment of angiogenesis in MM, and demonstrated benefits ofnanomedicine, the advantages nanoparticle based therapeutics can provideis yet to be harnessed to its full potential in MM.

PEGylated micellar nanoparticles offer a combination of increasedstability, high circulation times, and a defined size range of 10-100 nmfor increased tumor accumulation and decreased systemic toxicity. Animportant feature of micellar nanoparticles is that they presentparticularly attractive scaffolds for multivalent display as they can bedesigned to have multiple functional groups on their surfaces to presenttargeting moieties and drug conjugates simultaneously.

In the examples below, doxorubicin was conjugated to the nanoparticlesthrough an acid-sensitive hydrazone bond to prevent premature releaseand thus non-specific toxicity. Peptides were conjugated via amultifaceted synthetic procedure for generating precisely controllednumber of targeting functionalities per nanoparticle. The nanoparticlesexhibited a size of ˜20 nm, were efficiently internalized by MM cells,and induced cytotoxicity against MM cells. Mechanistic studies revealedthat nanoparticles induced DNA double strand breaks and inducedapoptosis, associated with H2AX phosphorylation, PARP and caspase-8cleavage. Importantly, multifunctional nanoparticles were moreefficacious than doxorubicin in the presence of fibronectin(IC50=0.15±0.04 μM and 0.42±0.09 μM, respectively), and overcame CAM-DRinduced by adherence of MM cells to fibronectin. Finally, in a MMxenograft model, nanoparticles preferentially homed to MM tumors, with a˜10 fold more drug accumulation when compared to doxorubicin, anddemonstrated dramatic tumor growth inhibition with much reduced overallsystemic toxicity.

Thus, the disease-driven engineering of a nanoparticle-based drugdelivery system may enable an integrative approach in the treatment ofMM. Described below are specific examples of self-assemblingmultifunctional micellar nanoparticles for targeted delivery of Dox toMM cells while overcoming CAM-DR. This is accomplished by designingparticles that are simultaneously functionalized with controlled numbersof VLA-4-antagonist peptides and pH sensitive Dox conjugates. When thenanoparticles are delivered, first they target VLA-4 expressing MM cellsand inhibit cellular adhesion via VLA-4, thereby overcoming CAM-DR. Atthe same time, nanoparticle binding to VLA-4 triggers receptor-mediateduptake, upon which active Dox is released due to pH-sensitive bondhydrolysis in the acidic endocytic vehicles. These examples demonstratethe disease-driven engineering of a nanoparticle-based drug deliverysystem, enabling the model of an integrative approach in the treatmentof MM.

EXAMPLE 1 Identification of a VLA-4 Antagonist Peptide That SelectivelyBinds to MM Cells and Inhibits MM Cell Adhesion to Fibronectin

MM cells express VLA-4 (α4β1 integrin) receptor that facilitatesadhesion of MM cells to the extracellular matrix protein fibronectin,thereby actuating CAM-DR development in MM cells. In several MM celllines, including NCl-H929, IM9, U266, and MM.1S, VLA-4 expression wasvalidated by detecting the expression of α4- and β1-subunits using flowcytometry. Both subunits were expressed on all cell lines tested (FIG.5A).

VLA-4 is a cell surface receptor that plays a critical role in cancersas well as autoimmune diseases, and several VLA-4 targeting peptideshave been identified. None of these peptides, however, have been testedfor their specific binding to MM cells, or their antagonistic effectsfor inhibiting cellular adhesion. Since both these criteria are crucialin this targeting strategy, a small library of peptides was generatedand screened from the literature.

For cellular binding assays we synthesized FITC labeled version of thepeptides and compared their affinity to MM cells by flow cytometry.

VLA-4-pep binds to MM cells and inhibits their adhesion to fibronectin.As shown in FIGS. 5a-5b , MM.1S, NCl-H929, U266, and IM9 cell lines allexpress VLA-4 subunits α4- and β1-integrins as determined by flowcytometry. Black columns are primary antibodies, and grey columns areisotype controls. FIG. 6a shows the results of cellular binding assaysperformed using FITC-labeled VLA-4-pep and detected by flow cytometry.Control experiments were done with FITC-labeled non-specific peptide andthe background binding was subtracted for each data point. VLA-4-pepbinds to U266, NCl-H929, and 1M-9 cell lines with apparent Kd of ˜250nM. As shown in FIG. 6b , VLA-4-pep inhibits adhesion of MM cell linesto fibronectin-coated plates. BSA coated plates were used as controls,and no adhesion of MM cells was observed. No inhibition of adhesion wasobserved in the control experiments done with non-specific peptide(results not shown). All experiments were done in triplicates and datarepresents means (±SD).

We found that peptide-[9] (VLA-4-pep; SEQ ID NO: 1; FIG. 4) binds to MMcell lines with specificity (FIG. 6a ). Control experiments done withFITC labeled non-specific peptide showed only minimal backgroundbinding, and was subtracted for each data point. Competition experimentsdone with excess unlabelled VLA-4-pep showed inhibition of fluorescencesignal, indicating that VLA-4-pep specifically binds to VLA-4 receptoron MM cells (results not shown). VLA-4-pep also prevailed as the mostpotent inhibitor of MM cell adhesion to fibronectin in a typicalcalcein-based cell adhesion assay (FIG. 5b ). Control experiments donewith non-specific peptide did not show any adhesion inhibitory effects(results not shown). Therefore, in our hands, VLA-4-pep prevailed asleading VLA-4 antagonist peptide and was incorporated as the targetingmoiety in the nanoparticles.

Another of the candidates we tested (the peptide sequence CFLDFP) had aweaker affinity for VLA-4. This sequence could be substituted forVLA-4-pep as a targeting molecule in nanoparticles for treating MM.Peptide sequences based on the X-CDPC core structure, where X is avariable amino acid, could also be substituted for VLA-4-pep as atargeting molecule in nanoparticles for treating MM.

EXAMPLE 2 Synthesis of VLA-4-pep and Dox Conjugated Lipids andNanoparticles

Peptides, and peptide/DSPE-PEG2000 lipid conjugates with fluorescencemoieties were manually synthesized on solid support, rink amide resin,using Fmoc chemistry (peptide synthesis chemicals/reagents fromNovaBiochem, DSPE-PEG2000 from Avanti Lipids Inc.). Resin cleavages ofall peptide products were done by TFA, purification via RP-HPLC, andcharacterization by MALDI-TOF-MS. Peptide cyclization through disulfidebond formation between cysteine residues was performed in 1 mL DMF with20 μL DIEA by stirring for 8 hrs at room temperature. DPPE-GA,hydrazine, and diisopropylcarbodiimide were mixed in a vial and allowedto react for 4 hours at room temperature. Solvent and excess reactantswere removed via evaporation under vacuum. Product was re-dissolved inchloroform, mixed with Doxorubicin in methanol and coupled over 3 days.Final product was isolated via extraction, and characterized with MS.

The VLA-4 targeting peptide, VLA-4-pep, was incorporated on thenanoparticle for active targeting of MM cells. VLA-4-pep/DSPE-PEG2000conjugate was synthesized using a synthetic strategy that was developedusing solid support methodology. FIG. 7a is a schematic illustration ofthe multifaceted synthetic steps for peptide conjugation toDSPE-PEG2000-NH2 using this solid support methodology. VLA-4-pep wasfirst synthesized on the rink amide resin using Fmoc protocols, followedby reacting succinic anhydride at the N-terminal amine to generate acarboxylic acid group at the peptide terminus. This newly generatedcarboxylic acid group on the resin bound peptide was activated, andDSPE-PEG2000-Amine lipid was introduced in anhydrous DMF to promoteamide coupling. The peptide-PEG-lipid conjugate was cleaved from theresin using a TFA cocktail, purified via HPLC and characterized byMALDI-TOF-MS.

Dox/DPPE-GA lipid conjugation was accomplished using a pH sensitivehydrozone chemistry to provide controlled drug release. FIG. 7b is aschematic illustration of Dox conjugation to DPPE-GA.

Nanoparticles were synthesized from the lipid-PEG block co-polymer,DSPE-PEG2000. This PEG-lipid, when placed in water, self-assembles toform micelles with 16 nm diameter. Their size stipulates the EPR effect,and prevents their entry through healthy endothelium pores. Meanwhile,the PEG conjugation increases micellar solubility and biocompatibility,provides the nanoparticles with stealth against the reticuloendothelialsystem and increases their circulation time. DSPE-PEG2000 lipid has alow CMC (5-10 μM) allowing for experimentation at therapeuticallyrelevant concentrations without lipid dissociation. DSPE-PEG2000 alsohas a terminal primary amine allowing for easy conjugation of variousdifferent molecular moieties.

Multifunctional micelles were prepared by mixing DSPE-PEG2000,VLA-4-pep/DSPE-PEG2000 conjugate, and Dox/DPPE-GA conjugate at desiredmolar ratios in DCM, followed by solvent removal via evaporation. Themixture was then re-suspended in PBS, and stirred until clear Eachmicelle comprises ˜90 lipid molecules, and their relative monodispersityallows for incorporation of precise numbers of functionalized lipids perparticle to provide control over valency of targeting peptide and drugloading.

Besides VLA-4 targeting, Dox conjugated multifunctional nanoparticles(NPDox/VLA-4-pep), only Dox conjugated (NPDox), only VLA-4-pepconjugated (NPVLA-4-pep), Dox and non-specific peptide conjugated(NPDox/ns), non-specific-peptide conjugated (NPns), and barenanoparticles (NPbare) was prepared for control experiments. For imagingand cellular uptake experiments, lissamine rhodamine PE was incorporatedin the micelles during formation. In all experiments the total lipidconcentration was above CMC.

Particle size was observed using dynamic light scattering analysis viathe 90Plus Nanoparticle Size Analyzer (Brookhaven Instruments Corp.),using 658 nm light observed at a fixed angle of 90° at 20° C. Allsamples were centrifuged for 30 minutes before analysis to eliminatedust and larger aggregates. FIG. 7c illustrates dynamic light scattering(DLS) analysis of the nanoparticles. The DLS analysis established thatregardless of the number and type of functional moieties included, theparticles maintained their original size of ˜20 nm. VLA-4 targeting, Doxconjugated (NPDox/VLA-4-pep), only Dox conjugated (NPDox), onlyVLA-4-pep conjugated (NPDox/VLA-4-pep), Dox and non-specific peptideconjugated (NPDox/ns), nonspecific-peptide conjugated (NPns), and barenanoparticles (NPbare) all gave an average size distribution around 20nm (FIG. 7c ).

Dox was conjugated to the nanoparticles via an acid labile bond toprevent the premature release of the chemotherapeutic and thusnon-specific toxicity. Upon endocytosis of nanoparticles, the acidicenvironment of endosomes catalyzes the release of active Dox, providinglocalized delivery inside tumor cells. To analyze the Dox releasekinetics, Dox conjugated nanoparticles ([Dox]=34.5 μM) were prepared andrelease rates were analyzed at pH=7.4 PBS buffer, pH=5.5 acetate buffer,and 0.24N HCl. Amounts of free Dox for all buffers at different timepoints were measured using a Toyopearl AF-Amino-650M resin (Tosoh,Tokyo, Japan) packed column on Agilent series 1200 HPLC according toabsorbance at 477 nm. All data was normalized to total Dox released inHCl solution where hydrolysis is 100% within ˜5 minutes.

FIG. 7d illustrates the drug release profile of Dox from thenanoparticles at pH=5.5 and pH=7.4. Rate of hydrolysis was quantifiedvia HPLC, taking measurements at pre-determined time intervals andobserving the absorbance at wavelength of 477 nm. Data shown are from arepresentative experiment. The drug release profiles we observed in pH7.4 and pH 5.5 established that Dox is released from the nanoparticlespreferentially under acidic conditions (FIG. 7d ).

EXAMPLE 3 Cellular Uptake Studies of VLA-4 Targeting Nanoparticles

Next we evaluated whether VLA-4-pep functionalized nanoparticles weretaken up by VLA-4 expressing MM cells and determined the optimal peptidevalency per micelle for most efficient uptake profiles. All MM celllines were obtained from ATCC, and were cultured as previouslydescribed. Methods are described in detail below.

Detection of α4- and β1-integrin subunit expression: cells were stainedwith anti-CD49d(PE) or anti-CD29(FITC) antibodies (BD Biosciences).Isotype matched antibodies were used as negative controls. For detectionof apoptotic cells, cells were stained with Annexin V (FITC) antibody(BD Pharmingen). Cells were analyzed with Guava EasyCyte flow cytometer(Millipore).

Cell-binding assays: MM cells were incubated on ice, for 1 h, withFITC-labeled peptides in binding buffer (25 mM Tris, 150 mM NaCl, 1.5 mMMgCl₂, 1.5 mM MnCl₂, 5 mM glucose, 1.5 mM BSA). Cells were washed twicewith PBS and were analyzed with Guava EasyCyte flow cytometer(Millipore). FITC-labeled scrambled peptide was used as a non-specificcontrol, and was subtracted for each data point.

Adhesion Assays: These were performed using the Vybrant Cell AdhesionAssay Kit (Molecular Probes) according to the manufacturer'sinstructions. Briefly, calcein-labeled MM cells were added tofibronectin-coated 96 well plates (40 μg/ml) in adhesion buffer(RPMI-1640/2% FBS) for 2 h. To evaluate the adhesion inhibitory effectsof VLA-4-pep or VLA-4-pep functionalized nanoparticles, calcein labeledcells were added to fibronectin-coated plates, and immediately treatedwith the inhibitory agents. Non-adherent cells were removed by washingtwice with PBS. Adherent cells were quantitated in a fluorescencemulti-well plate reader.

Cellular Uptake studies: MM cells were incubated at 37° C. withrhodamine-labeled nanoparticles in complete media for the indicated timepoints and were analyzed with Guava EasyCyte flow cytometer. Forconfocal microscopy experiments, cytospin of nanoparticle-treated cellswere prepared on glass slides and fixed with 4% paraformaldehyde.Coverslips were then mounted on the glass slides with VectaShieldantifade/DAPI (Vector Labs). Cells were visualized by Nikon A1R confocalmicroscope with a 40× oil lens. Image acquisition was performed by NikonElements Ar software (Nikon).

Cellular uptake of rhodamine-labelled nanoparticles with varying numberof VLA-4-pep conjugates (n=0-40/nanoparticle) were studied via flowcytometry. FIGS. 8a-8c illustrate the results of cellular uptakestudies. Referring first to FIG. 8a , rhodamine-labeled nanoparticleswith varying valency of VLA-4-pep conjugates (n=0-40/nanoparticle) wereprepared and incubated with NCl-H929 cells at 37° C. for the indicatedtime points. Nanoparticle uptake by NC1-H929 cells increased withincreasing VLA-4-pep valency up to n=20, however dropped dramatically atn=40 (FIG. 8a ). Specifically, we observed that 20 peptides per particleyielded the maximum uptake, with up to 10 fold enhancement over that ofnon-targeted micelles (n=0) after 24 h.

In a separate experiment, we also used nanoparticles conjugated withnon-specific peptide (n=20) as a control, and observed similar resultsto that of non-targeted nanoparticles (FIG. 8b ). Control experimentswith non-specific peptide conjugated nanoparticles (NPns) andcompetition experiments with excess free VLA-4-pep (2 mM) were performedto determine receptor-mediated specificity of nanoparticle uptake. Datapresented in FIG. 8b represents means (±SD) of triplicate experiments.

To establish that uptake of VLA-4-pep conjugated particles werereceptor-mediated, we performed competition experiments, where MM cellswere co-incubated with VLA-4-pep conjugated nanoparticles (n=20) andexcess free VLA-4-pep. As shown in FIG. 8c , the internalization ofVLA-4 targeting nanoparticles was confirmed with a Nikon A1R confocalmicroscope using a 40× oil lens. Image acquisition was performed byNikon Elements AR software. The results showed a dramatic reduction incellular uptake back to the levels of non-targeted nanoparticles,proving receptor involvement in uptake (FIG. 8c ). It is noteworthy thatwe observed some nanoparticle uptake even with non-targeted micellesindicating low levels of non receptor-mediated uptake (FIGS. 8a,b ).

The studies described above were performed using flow cytometricanalysis, as it is a highly accurate quantitative method for studyingthe effect of peptide valency on uptake. One shortcoming of this method,however, is that it does not discriminate surface bound nanoparticlesfrom internalized ones. Therefore, to show that the nanoparticles areindeed internalized by MM cells, we performed confocal microscopyexperiments. These experiments revealed clear uptake of VLA-4-pepconjugated nanoparticles starting around 4 h and peaking at 24 h (FIG.8c ). Altogether, these studies showed efficient receptor-mediateduptake of nanoparticles with optimal uptake properties of n=20 VLA-4-pepper micelle. Therefore valency of 20 peptides per particle was used forrest of our studies.

EXAMPLE 4 Multifunctional Nanoparticles Are Cytotoxic to MM

We evaluated the cytotoxicity of NPDox/VLA-4-pep nanoparticles againstNCl-H929 MM cells using a colorimetric assay as follows.

Cytotoxicity assays: Cytotoxicity was determined using Cell CountingKft-8 (Dojindo) as previously described. To determine cytotoxicity inthe presence of fibronectin, MM cells were plated on fibronectin coatedplates (40 μg/ml), in adhesion buffer, for 1 h. Cells were then treatedwith increasing concentration of nanoparticles or free Dox (equivalentDox concentrations of NPDox/VLA-4-pep, NPDox, or free Dox) in completemedia with 10% FBS, for 48 h and 72 h. BSA coated plates were used ascontrols. In all cases, cell viability was assessed by cell countingkit-8 (CCK-8), and data represent means (±SD) of triplicate cultures.The results are shown in FIG. 9.

NPDox/VLA-4-pep was significantly cytotoxic to MM cells with IC50 valuesof 0.39±0.06 μM (48 h), and 0.13±0.02 μM, at 48 and 72 h, respectively(FIG. 9). Control experiments performed with equivalent doses of freeDox showed a moderate advantage to NPDox/VLA-4-pep at 48 h(IC₅₀=0.19±0.04 μM). This difference was diminished at 72 h, and bothfree Dox and NPDox/VLA-4-pep showed similar cytotoxic effects(IC₅₀=˜0.13 μM). The difference in cytotoxicity at 48 h is expectedgiven the differences in the cellular uptake mechanisms of free Dox andNPDox/VLA-4-pep. While free Dox is taken up via passive diffusion and isactive immediately, we designed our nanoparticles to release active Doxonly after they are internalized and are exposed to the acidicenvironment of the endocytic vesicles. These differences would lead tothe temporal difference observed in cytotoxicity. Control experimentsdone with non-targeted NPDox showed much reduced cytotoxic effects at 48and 72 h, further confirming VLA-4's role in nanoparticle uptake (FIG.9). Control experiments done with nanoparticles conjugated withnon-specific peptide (NPDox/ns) yielded very similar results to thoseobtained with non-targeted NPDox (results not shown). Controlexperiments performed with NPDox/ns showed similar results to NPDox andNPbare, and NPVLA-4-pep did not show any cytotoxic effects at theconcentrations tested (results not shown). No cytotoxic effects wereobserved in additional control experiments performed with nanoparticleslacking Dox conjugates, such as NPbare or NPVLA-4-pep, at equimolarparticle concentrations (results not shown).

EXAMPLE 5 Multifunctional Nanoparticles Induce DNA Double Strand Breaks(DSBs) and Apoptosis in MM Cells

It is well established that Dox induces DNA double strand breaks (DSBs)and apoptosis of cancer cells. An early specific cellular response toDSBs in mammalian cells is the phosphorylation of the histone proteinH2AX (γ-H2AX), with respective foci formation. Therefore, we examinedwhether free Dox and NPDox/VLA-4-pep triggered similar signalingcascades of DNA damage and apoptosis in MM cells. NCl-H929 cells weretreated with 250 nM Dox equivalents of NPDox/VLA-4-pep or free Dox for0-48 h. Phosphorylation of DNA damage response protein H2AX at Ser139was assayed by western blotting (FIG. 10a ). Respective H2AX fociformation was assayed by immunocytochemistry (FIG. 10b ). Apoptosis wasassessed by flow cytometry following Annexin V-FITC staining (FIG. 10c), and by western blotting for PARP cleavage and caspase-8 and caspase-9activation (FIG. 10d ).

For immunocytochemistry analysis, cytospins of drug-treated cells wereprepared on glass slides and fixed with 4% paraformaldehyde. Slides wereblocked with 5% goat serum, incubated with y-H2AX antibody (CellSignaling) overnight at 4° C., and then with AlexaFluor-488-labelledFab2 (Molecular Probes). Coverslips were then mounted on the glassslides with VectaShield antifade/DAR (Vector Labs) and analyzed by NikonEclipse TS100 fluorescence microscope at 60×/0.5-1.25 oil, with a NikonInfinity camera. For flow cytometric analysis, data represent means(±SD) of triplicate experiments. For western blotting, representativeimages are shown, and all antibodies were purchased from Cell Signaling.

Western blot and immunocytochemical analysis showed that both free Doxand NPDox/VLA-4-pep induced H2AX phosphorylation in NCl-H929 cells(FIGS. 10a, b ). Furthermore, both agents induced apoptosis as wasdetected by flow cytometric analysis of the early apoptotic markerannexin V (FIG. 10c ), and western blot analysis of PARP and caspase-8activation (FIG. 10d ). No significant caspase-9 activation was detectedby either agent. Altogether, these results suggest that free Dox andNPDOX/VLA-4-pep exert their cytotoxic effects through the similarcytotoxic mechanisms. It is noteworthy that no cell death or caspaseactivation was detected before 36 h at these doses. Therefore, formationof DSBs was not a secondary event of apoptosis.

EXAMPLE 6 Multifunctional Nanoparticles Inhibit Adhesion of MM Cells toFibronectin & Overcome CAM-DR

VLA-4-pep serves two major purposes in our nanoparticle design: i)selective targeting of VLA-4 expressing MM cells, and ii) inhibition ofMM cell adhesion to the stroma to overcome CAM-DR. To test whetherNPDox/VLA-4-pep overcame CAM-DR, first we evaluated its efficiency ininhibiting MM cells' adhesion to fibronectin. Calcein-labeled NCl-H929MM cells were allowed to adhere to fibronectin coated plates alone, orwith increasing concentrations of NPVLA-4-pep. Non-adherent cells wereremoved by washing with PBS, and adherent cells were quantitated in afluorescence multi-well plate reader. Data represents means (±SD) oftriplicate experiments. *P<0.05, **P<0.01 when compared to control. Doxwas not incorporated to the nanoparticles during this assay to eliminatecompounding effects that would result from cell death. No inhibition ofadhesion was observed with NPbare or NPns (results not shown).NPVLA-4-pep inhibited adhesion of calcein-loaded NCl-H929 cells tofibronectin in a dose dependent manner (FIG. 11a ).

Next, we compared the cytotoxic effects of free Dox and NPDox/VLA-4-pepagainst MM cells in the presence or absence of fibronectin. NCl-H929cells were allowed to adhere to fibronectin or BSA coated plates for 1h, and then treated with equivalent Dox concentrations ofNPDox/VLA-4-pep, or free Dox for 72 h. Fibronectin coated plates wereused to allow for adhesion of NCl-H929 cells, and BSA coated plates wereused for culturing cells in suspension (MM cells do not adhere to BSAcoated plates). Cell viability was assessed by cell counting kit-8(CCK-8), and data represent means (±SD) of triplicate cultures. Adhesionof NCl-H929 cells to fibronectin caused CAM-DR in the free Dox treatmentgroup with a 3-fold IC50 shift from 0.13±0.04 μM to 0.42±0.09 μM (FIG.11b , left). In the NPDox/VLA-4-pep treatment group, however, the IC₅₀values merged towards ˜0.2 μM both for the adherent and suspension MMcells, indicating that NPDox/VLA-4-pep overcame CAM-DR (FIG. 11b ,right).

The significance of these findings is best illustrated in FIG. 11c ,which shows an alternative view of data presented in FIG. 11b . When MMcells are cultured in suspension, the efficacy of free Dox in cellkilling is similar to that of NPDox/VLA-4-pep with an IC₅₀=˜0.13 μM(FIG. 11c , left). On the other hand, when the cells are cultured in thepresence of fibronectin, NPDox/VLA-4-pep (IC₅₀=0.15±0.04 μM) is moreefficacious than free Dox (IC₅₀=0.421−0.09 μM). These results suggestthat NPDox/VLA-4-pep overcomes CAM-DR in MM cells.

EXAMPLE 7 Multifunctional Nanoparticles Preferentially Home to MM Tumorsand Inhibit Tumor Growth in Vivo

FIGS. 12a-12f illustrate the in vivo characterization of NPDox/VLA-4-pepin a subcutaneous xenograft model of MM. To validate the therapeuticefficacy of multifunctional nanoparticles, CB.17 SCID mice (HarlanLaboratories) were irradiated with 150 rad, and were inoculatedsubcutaneously with 5×10⁶ NCl-H929 cells. The SCID mice were randomlydistributed into 4 treatment groups of 8 mice: i) free Dox (6 mg/kg),ii) NPDox/VLA-4-pep (6 mg/kg Dox equivalent), iii) NPDox (6 mg/kg Doxequivalent), and iv) PBS (vehicle control). When the tumors werepalpable, each mouse was injected with the drug, intravenously, on days1, 3, and 5. Tumor bearing SCID mice were injected, iv) with free Dox,NPDox/VLA-4-pep, or NPDox, at a dose of 6 mg/kg Dox equivalents on days1, 3 and 5. Animals were monitored for body weight and tumor volume bycaliper measurements. Tumor growth inhibition was detected by calipermeasurements (left). Data shown are means (±SE) of n=6-8 per treatmentgroup. Statistical comparisons of continuous variables were carried outby Student's two-tailed t-test and were considered significant whenP<0.05.

For determination of systemic toxicity, 3 additional mice from eachgroup were sacrificed on day 5 before any lethality was observed. Organweights were measured. For complete blood count analysis, 200 μl ofblood was drawn from each mouse via cardiac puncture, immediately mixedwith 50 μl of Sequester Solution (Cambridge Diagnostic Products), andwas analyzed with the HemaVet950 (Drew Scientific). Immunohistochemicalstaining of excised tumors for caspase-3 was performed as previouslydescribed.

The results are shown in FIGS. 12a-12f . As indicated, both free Dox andNPDox/VLA-4-pep resulted in dramatic tumor growth inhibition (FIG. 12A).However, at the dose used, free Dox resulted in a significant loss ofbody weight, and caused lethality of all mice on day 7 because of highsystemic toxicity (FIG. 12b , % body weight of animals as a measure ofsystemic toxicity). Only ≤15% loss was observed with NPDox/VLA-4-pep orNPDox. The NPDox/VLA-4-pep group only lost ≤15% body weight and nolethality was observed during the duration of the study (FIG. 12b ).NPDox/VLA-4-pep was significantly more efficacious than NPDox with*P<0.05 (FIG. 12a , right). These results indicate that NPDox/VLA-4-pephas a much-improved therapeutic index when compared to free Dox. NPDoxalso showed tumor growth inhibition, but was significantly lessefficacious than NPDox/VLA-4-pep (FIG. 12a , right).

Three mice from each group were dissected on day 5 and tumors werestained for activated caspase-3. Representative images of tumorcross-sections that were captured using a Nikon Eclipse TS100 microscopeat 20× magnification are shown. Ex-vivo mechanistic studies performed ontumors dissected on day 5 showed that all drug treatment groups inducedapoptosis associated with caspase-3 activation (FIG. 12c ).

NPDox/VLA-4-pep can expectedly accumulate in the tumor through theenhanced permeation and retention (EPR), as well as the VLA-4 targetingfunctionality, resulting in reduced systemic toxicity. To evaluateenhanced tumor uptake, the tissue biodistribution of Dox was studied forall treatment groups. Three mice per group were dissected 24 h afterinjection with 10 mg/kg free Dox or Dox equivalent nanoparticles,processed as previously described, and analyzed by fluorescencespectroscopy for Dox fluorescence (ex. 490 nm/em. 550 nm).

Tissue biodistribution of Dox following treatment is shown in FIG. 12d .Data shown are means (±SE). *P<0.05, **P<0.01 when compared to free Doxgroup. No significant difference was detectable in the distribution ofDox in lung, kidney, heart, or spleen at 24 h, however, a very highaccumulation of Dox was observed in the tumor for the NPDox/VLA-4-pepgroup when compared to free Dox and NPDox, reaching to ˜10 and ˜5 foldhigher levels, respectively (FIG. 12d ). These results are consistentwith the enhanced tumor growth inhibition observed with NPDox/VLA-4-pepand demonstrate that incorporating VLA-4-pep to the nanoparticlesenabled enhanced targeting of VLA-4 expressing MM tumors. Doxaccumulation in the tumor was ˜2 fold more for NPDox, when compared tofree Dox.

To evaluate systemic toxicity, analysis of complete blood cell count,which is highly susceptible to chemotherapeutic agents, was performed.Three mice from each group were dissected on day 5, and complete bloodcount (white blood cell, red blood cell, and thrombocyte) was performed(FIG. 12e ). Weights of excised heart, kidney, spleen and liver areshown in FIG. 12e . Data represents means (±SE). *P<0.05, when comparedto free Dox group.

Systemic toxicity was detectable in all treatment groups as evident fromwhite blood cell (wbc) red blood cell (rbc), and thrombocyte counts(FIG. 12e ). NPDox/VLA-4-pep group, however, showed significantly lesstoxicity on wbc and thrombocyte counts when compared to free Dox (FIG.12e ).

Dox has been associated with clinically significant cardiac and renaltoxicity. The effect of the nanoparticles on cardiac and renal weightloss was therefore evaluated. All treatment groups showed a mildreduction in cardiac mass, with no detectable difference betweenNPDox/VLA-4-pep and free Dex (FIG. 12f ). Previous studies performed inanimal models have shown that free Dox is significantly more toxic tocardiac tissue when compared to nanoparticles. The lack of such adifferential in the results could be due to the relatively early timepoint (5 days) at which the analysis was performed. On the other hand, asignificant difference on kidneys was detected, as NPDox/VLA-4-pep wassignificantly less toxic to the kidneys than free Dox and did not causeany significant renal mass loss (FIG. 12f ).

It is noteworthy that, based on biodistribution studies, significant Doxaccumulation was evident in kidneys in all treatment groups (FIG. 12d ).It is possible that the reduced toxicity of the nanoparticles on kidneysis due to the acid sensitive hydrazone bond, which releases active Doxonly after receptor-mediated uptake, or in the acidic microenvironmentof the tumor tissue.

Nanoparticles are known to accumulate in and be cleared by thereticuloendothelial system (RES) organs (spleen/liver). Therefore, theeffect of nanoparticles on spleen and liver was analyzed. All treatmentgroups, including free Dox, showed significant and similar accumulationin spleen (FIG. 12f ) and severe mass loss (FIG. 12f ).Histopathological examination of spleen revealed severe hypoplasia ofboth erythroid and myeloid elements in all drug treatment groups.Nanoparticles, however, showed only moderate fibrosis, whereas severefibrosis was evident in the free Dox group. Although nanoparticles of˜100 nm diameter have been shown to differentially accumulate in spleen,the small size of our micellar nanoparticles (˜20 nm) may provide amechanism to escape the spleen compartment of RES, leading to similaraccumulation and toxicity to free Dox. On the other hand,biodistribution studies suggested an increased accumulation ofnanoparticles in liver (FIG. 12d ), but with reduced mass loss than freeDox (FIG. 12f ). Histopathological analysis of liver revealed moderatehepatocellular hypertrophy and degeneration in the free Dox group,whereas only mild effects were observed in the nanoparticle treatmentgroups. Increased accumulation to liver without increased toxicity wasalso shown in previous studies and could be due to the acid sensitivehydrazone bond, which requires receptor-mediated uptake or the acidicmicroenvironment of the tumor cells to release active Dox. Altogetherthese results indicate that NPDox/VLA-4-pep showed decreased overallsystemic toxicity than free Dox.

In summary, in vivo studies demonstrated dramatic tumor growthinhibition, significantly increased accumulation in the tumor, andoverall decreased systemic toxicity by NPDox/VLA-4-pep. Combined, theresults suggest improved therapeutic index for NPDox/VLA-4-pep incomparison to free Dox.

In this study, multifunctional micellar nanoparticles were engineeredthat target VLA-4 expressing MM cells selectively, while combiningcellular adhesion inhibitory effects and cytotoxic effects in a temporalfashion to overcome CAM-DR. In our design, peptides were used astargeting agents, which have several advantages over antibodies such asfavorable pharmacokinetics, easy derivatizing and manufacturing, andlower cost. Another advantage is that, unlike antibody therapeutics thatrely on high affinity interactions, peptides can be selected to have abroad range of binding affinities. In physiological systems, multiplelow affinity interactions are used to distinguish one cell type fromanother and to provide selectivity. Multivalent presentation improvesnot only the binding affinity but also the specificity ofligand-receptor interactions.

A low affinity VLA-4 antagonistic peptide was selected for these studies(K_(d) ˜0.25 μM; FIG. 4), and micellar nanoparticles were used asdynamic self-assembling scaffolds to multivalently present this peptideto target VLA-4 overexpressing MM cells. Receptor-mediated endocytosisis a particularly important aspect in the design of the above-describedexamples of nanoparticles, since the acidic environment of the endocyticvesicles are required for active Dox release (FIG. 7d ). The resultsdemonstrated that binding of the nanoparticles to VLA-4 triggeredreceptor-mediated uptake. Interestingly, it was observed that theefficiency of uptake depended on the number of targeting peptides permicelle, with an optimal number of 20 peptides per micelle. While theoptimal number of targeting peptides per micelle may vary based on thepeptide's monovalent affinity, as well as its k_(on) and k_(off) rateconstants, these studies validated VLA-4 as a suitable target fortargeted drug delivery in MM.

One of the key findings of the study was the efficacy of NPDox/VLA-4-pepon MM cell cytotoxicity in the presence of the extracellular matrixprotein fibronectin. When the cells are cultured in suspension, in theabsence of fibronectin, the efficacy of free Dox in cell killing wassimilar to that of NPDox/VLA-4-pep. However, when the cells were allowedto adhere to fibronectin, NPDox/VLA-4-pep proved to be more efficaciousthan free Dox (FIG. 11). In other words, while adhesion of MM cells tofibronectin provided CAM-DR when cells were treated with free Dox,NPDox/VLA-4-pep significantly overcame CAM-DR. These results establishthe significance of targeting MM cells as well as their interactionswith the microenvironment in the design of more effective noveltherapeutics.

Several different mouse models of MM were described. Here, asubcutaneous xenograft model of MM was used for various advantages thismodel provides, such as the formation of palpable tumors, which makestumor growth inhibition and biodistribution studies feasible. Inaddition, tumors can be excised to enable ex-vivo mechanistic analysis.A key factor in drug delivery efficiency is the tumor-to-normal organuptake ratios of the chemotherapeutic agents. The in vivo resultsdemonstrated that NPDox/VLA-4-pep preferentially accumulates in thetumor when compared to free Dox and NPDox. Most importantly,NPDox/VLA-4-pep showed dramatic tumor growth inhibition with decreasedoverall systemic toxicity, demonstrating improved therapeutic index. Itis noteworthy that VLA-4-pep targets human VLA-4, and thatNPDox/VLA-4-pep may have a different toxicity profile in humans.

One shortcoming of the subcutaneous xenograft model is the growth oftumors in the lack of BM microenvironment. Therefore the growth andsurvival advantages provided by the microenvironment are not wellrecapitulated in this model. Given that the inhibition of CAM-DR effectof NPDox/VLM4-pep is best emphasized in the presence of the BMmicroenvironmental factors such as fibronectin (FIG. 11), theimprovement of efficacy observed with NP Dox/VLA-4-pep using this modelmay be an underestimate. Thus, dosages and timing of administration ofnanoparticles as described herein may be determined using known methods,and may vary among embodiments.

In summary, nanotechnology has been harnessed to develop a “one stone,two bird” combinational therapy approach for MM, where Dox conjugatednanoparticles selectively targeted VLA-4 expressing MM cells, preventeddevelopment of CAM-DR, and dramatically inhibited tumor growth withoverall reduced systemic toxicity. Taken together, this study providesthe preclinical rationale for the clinical evaluation of VLA-4targeting, Dox conjugated multifunctional nanoparticles to improvepatient outcome.

In addition to the advantages offered by including both a targetingmoiety and a therapeutic agent, the present disclosure describes methodsfor constructing and designing nanoparticles. In contrast to previousmethods, the methods described herein may be used to producenanoparticles with consistent pre-determined molecular ratios of theincluded components, and less variation in nanoparticle size andcomposition both within batches and between batches. For example,methods described herein may be used to produce nanoparticles with adesired size (e.g., nanoparticles with 80-100 molecules, or 20-40 nm indiameter), a desired targeting molecule valence (e.g., 15-30 targetingmolecules, or about 20 targeting molecules), and/or a desired molecularratio of components.

FIG. 13 illustrates a flow diagram of a method 1300 for constructing adrug delivery nanoparticle, in accordance with various embodiments.

Optionally, method 1300 may begin at block 1301 with the synthesis ofone or more targeting molecules (e.g., targeting molecule 140). Atargeting molecule may be, or may include, a peptide, a cyclic peptide,a peptidomimetic, and/or a small molecule. Optionally, the targetingmolecule may be an antagonist of a target cell receptor. The targetingmolecule may be configured to bind to a target cell, target tissue, orcomponent of extracellular matrix (ECM). In some embodiments, thetargeting molecule may be configured to bind to a surface molecule(e.g., an integrin, a cadherin, a selectin, or syndecan) of a targetcell. As described in the examples above, the targeting molecule may beconfigured to trigger endocytosis by the target cell. The targetingmolecule may be configured to bind to a surface molecule of a targetcell with low affinity. Optionally, the targeting molecule may beconfigured to reduce or disrupt adhesion of a target cell to anothercell or ECM. In a specific example, the targeting molecule may be aVLA-4 antagonist peptide, such as VLA-4-pep (SEQ ID NO: 1).

In some embodiments, method 1300 may further include selecting atargeting molecule. For example, a molecular modeling program may beused to generate a list of possible candidates for targeting a moleculeon the target cell, such as a cell surface receptor (e.g., VLA-4). Thecandidates may be synthesized and screened using binding assays such astitrations, flow cytometry, and/or ELISA assays. One or more of thecandidates may be selected for use as targeting molecules based on thebinding affinity, the chemistry of conjugation, and/or various molecularproperties. In some embodiments, a candidate may be selected for use asa targeting molecule based at least in part on having a binding affinity(to a target cell surface receptor) in the range of 1 nM to 1 μM. Thus,in some embodiments, a targeting molecule may have a binding affinity inthe range of 1 nM to 1 μM. Alternatively, the targeting molecules mayhave a binding affinity in the range of 1 nM to 0.5 μM, 0.1 μM to 0.4 μ,0.2 μM to 0.7 μM, or 0.2 μM to 0.3 μM. In embodiments that include theuse of the targeting molecule conjugation methodology described in theexamples above, candidates may be selected for use as targeting moleculebased at least in part on a lack of secondary structure, as theabove-described conjugation chemistry will denature the molecule.Optionally, a candidate may be selected for use as a targeting moleculebased at least in part on amenability to a solid phase synthetic design.

At block 1303, a plurality of the targeting molecules may be coupled toa corresponding plurality of first lipid molecules to form a firstcomponent. In some embodiments, the targeting molecules may beconjugated to the lipid molecules. In some embodiments, the firstcomponent may further include a polymer that is conjugated or otherwisecoupled to a lipid molecule. The polymer may be a relativelywater-soluble polymer, such as polyethylene glycol (PEG).

In one embodiment, the first component may include a targeting moleculecoupled to a lipid-PEG block copolymer (e.g., VLA-4-pep coupled toDSPE-PEG2000). Optionally, the targeting molecule may be coupled to afirst end of the polymer, and the lipid molecule may be coupled to anopposite second end of the polymer. The targeting molecule may besynthesized on amide resin and reacted with succinic anhydride togenerate a carboxylic acid group at the peptide terminus. Thiscarboxylic acid group may then be activated, and DSPE-PEG2000-Aminelipid may be introduced in anhydrous DMF to promote amide couplingbefore cleaving the peptide-PEG-lipid conjugate from the resin with anacid (e.g., a TFA cocktail).

Optionally, at block 1305, a plurality of therapeutic agent moleculesmay be coupled to a corresponding plurality of second lipid molecules toform a second component. The second lipid molecules may be lipids of adifferent species/structure than the first lipid molecules.Alternatively, the second lipid molecules may be lipids of the samespecies/structure as the first lipid molecules. Some or all of thetherapeutic agent molecules may be coupled to the corresponding secondlipid molecules by a pH-sensitive bond. For example, some or all of thetherapeutic agent molecules may be coupled to the corresponding lipidmolecules by an acid-labile bond, such as (but not limited to) ahydrazone (HN-NH) bond. The therapeutic agent molecules can be orinclude any type of molecules for which delivery to a target cell ortissue is desired. In one example, the therapeutic agent is doxorubicin.In other examples, the therapeutic agent may be another drug (e.g., achemotherapy drug for treating a cancer), a nucleic acid (e.g., siRNA,DNA, etc.), or some combination thereof. Optionally, some of themolecules of the second lipid may be coupled to corresponding moleculesof a first therapeutic agent, and other molecules of the second (orother) lipid may be coupled to corresponding molecules of a secondtherapeutic agent.

In a specific example, the lipid may be DPPE-GA and the therapeuticagent may be doxorubicin. The DPPE-GA may be coupled to the doxorubicinmy mixing the DPPE-GA with hydrazine and diisopropylcarbodiimide,allowing these reagents to react (e.g., for 4 hours at roomtemperature), removing solvent and excess reactants (e.g., viaevaporation under vacuum), re-dissolving the product in chloroform andmixing with doxorubicin in methanol, and allowing these reagents tocouple over a period of time (e.g., 3 days).

At block 1307, the first and second components and a third component maybe added to a first solvent in a predetermined molar ratio. Thepredetermined molar ratios of the first, second, and third componentsmay vary among embodiments. The predetermined molar ratio may be afunction of the desired molar percentages and/or molecular ratios of thecomponents in the nanoparticles, discussed above. For example, toconstruct a nanoparticle with a desired molecular ratio of 60:20:10(third component:first component:second component), the components maybe added in a predetermined molar ratio of 6:2:1. The molar percentageof the first component (i.e., targeting component) may be 0-60% (e.g.,0-5%, 0-40%, 1-40%, 10-30%, 15-35%, 20-30%, 20-40%, or 30-40%). Themolar percentage of the second component (i.e., therapeutic component)may be 0-25% (e.g., 0-5%, 1-10%, 0-20% 1-20%, 5-15%, 5-20%, or 10-20%).The molar percentage of the third component may be 60-100% (e.g.,60-70%, 60-90%, 70-90%, 75-95%, 80-90%, 80-95%, or 90-100%). In someembodiments, the molar percentage of the third component may be greaterthan the molar percentage of the first component, which may be greaterthan the molar percentage of the second component. Alternatively, themolar percentage of the second component may be greater than the molarpercentage of the first component.

The ratio of the second component to the first and third components maybe selected to result in nanoparticles with a desired number of thetargeting molecules for multivalent presentation to the target cell andenhanced binding affinity and/or specificity of ligand-receptorinteractions. The third component may be configured to enhance thestability and/or circulation time of a nanoparticle. In someembodiments, the first component may include a polymer that isconjugated or otherwise coupled to a lipid molecule. The polymer may bea relatively water-soluble polymer, such as polyethylene glycol (PEG).In some embodiments, the third component may be a lipid-PEG blockcopolymer (e.g., DSPE-PEG2000). Optionally, two or more of the first,second, and third components may include the same lipid and/or polymerspecies. For example, the first component and the third component mayinclude a lipid-PEG block copolymer (e.g., DSPE-PEG2000).

In a particular embodiment, the first component is apeptide-PEG-phospholipid conjugate (e.g., VLA-4-pep/DSPE-PEG2000), thesecond component is a therapeutic agent conjugated to a phospholipid(e.g., Dox/DPPE-GA), and the third component is a pegylated phospholipid(e.g., DSPE-PEG2000). In this embodiment, the constructed nanoparticlesmay include 80-100 component molecules, or about 90 component molecules.The predetermined molecular ratio of third component:firstcomponent:second component may be 60:20:10. The molar percentages of thethird, first, and second components may be about 67%, about 22%, andabout 11%, respectively.

At block 1309, the first, second, and third components may be mixed inthe first solvent to form a plurality of nanoparticles that include thecomponents in the predetermined molar ratio. The resulting nanoparticlesmay have diameters in the range of 18-90 nm, 20-50 nm, 20-40 nm, 18-30nm, or 18-25 nm. Optionally, the first solvent may be removed (e.g., byevaporation) and the nanoparticles may be resuspended in an aqueoussolution. In some embodiments, the resulting nanoparticles may bemicelles comprising 80-100 molecules, collectively, of the first,second, and third components. In one embodiment, 10-40 of the 80-100molecules may be molecules of the first component. In a particularembodiment, the nanoparticles may have a diameter of 18-30 nm and atotal number of 80-100 molecules, of which 15-25 molecules are moleculesof the first component. In still other embodiments, the resultingnanoparticles may be micelles comprising more than 100 molecules or lessthan 80 molecules.

FIG. 14 illustrates a flow diagram of a method 1400 for designing andusing a drug delivery nanoparticle to treat a target cell or tissue, inaccordance with various embodiments.

Method 1400 may begin at block 1401 with the selection of a target cellor tissue for treatment with a therapeutic agent. The target cell ortissue may be, for example, a cancerous cell or tissue (e.g., a solidtumor, a blood cell cancer, etc.). The target cell or tissue may have afirst surface receptor that is not present on, or is present in fewernumbers or in a different arrangement on, a non-target cell or tissue.

At block 1403, a ligand that binds to the first surface receptor may beselected. In some embodiments, this may be accomplished by screening avariety of potential ligands and selecting one based on bindingaffinity. In some embodiments, a molecular modeling program may be usedto generate a list of possible candidates for targeting a molecule onthe target cell, such as a cell surface receptor (e.g., VLA-4). Somecandidates may be molecules known in the art for binding to a targetcell surface receptor (e.g., known antagonists of a receptor).Optionally, some or all of the candidates may be synthesized. Candidatesmay be screened using binding assays such as titrations, flow cytometry,and/or ELISA assays. One or more of the candidates may be selected foruse as targeting molecules based on the binding affinity, the chemistryof conjugation, and/or various molecular properties. In someembodiments, a candidate may be selected for use as a targeting moleculebased at least in part on a binding affinity (to a target cell surfacereceptor) of 1 nM to 1 μM. In embodiments that include the use of thetargeting molecule conjugation methodology described in the examplesabove, candidates may be selected for use as targeting molecule based atleast in part on a lack of secondary structure, as the above-describedconjugation chemistry will denature the molecule. Optionally, acandidate may be selected for use as a targeting molecule based at leastin part on amenability to a solid phase synthetic design.

Optionally, the targeting molecule may be configured to bind to asurface molecule of a target cell and/or ECM component with lowaffinity. The targeting molecule may be configured to triggerendocytosis by the target cell upon binding to the surface molecule.Optionally, the targeting molecule may be configured to reduce ordisrupt adhesion of a target cell to another cell or ECM. In a specificexample, the targeting molecule may be a VLA-4 antagonist peptide, suchas VLA-4-pep (SEQ ID NO: 1).

At block 1405, molecules of the selected ligand may be coupled tocorresponding molecules of a first lipid molecule to form a firstcomponent. In some embodiments, the ligand may be conjugated to thelipid molecules. In some embodiments, the first component may furtherinclude a polymer that is conjugated or otherwise coupled to a lipidmolecule. The polymer may be a relatively water-soluble polymer, such aspolyethylene glycol (PEG). In a specific embodiment, the first componentmay include a ligand coupled to a lipid-PEG block copolymer (e.g.,VLA-4-pep coupled to DSPE-PEG2000). Optionally, the ligand may becoupled to a first end of the polymer, and the lipid molecule may becoupled to an opposite second end of the polymer.

At block 1407, the therapeutic agent may be coupled to a plurality ofsecond lipid molecules to form a second component. Some or all of thetherapeutic agent molecules may be coupled to the corresponding secondlipid molecules by a pH-sensitive bond, such as (but not limited to) ahydrazone (HN-NH) bond. The therapeutic agent can be or include any typeof drug or other agent for which delivery to a target cell or tissue isdesired. In one example, the therapeutic agent is doxorubicin. In otherexamples, the therapeutic agent may be another drug (e.g., achemotherapy drug for treating a cancer), a nucleic acid (e.g., siRNA,DNA, etc.), or some combination thereof.

At block 1409, nanoparticles may be formed by adding the first andsecond components and a third component to a first solvent in apredetermined molar ratio. In some embodiments, the mole percentagerange for the second component (i.e., therapeutic component) is 0-20%,the mole percentage range for the first component is 0-40%, and the molepercentage range for the third component is 60-100%. The resultingnanoparticles may have diameters and relative ratios of components asdescribed above. In some embodiments, the third component may be alipid-PEG block copolymer (e.g., DSPE-PEG2000). Optionally, two or moreof the first, second, and third components may include the same lipidand/or polymer species. For example, the first component and the thirdcomponent may include a lipid-PEG block copolymer (e.g., DSPE-PEG2000).

In some embodiments, several groups or batches of nanoparticles may beformed separately, each group having a different molar ratio of thefirst component to the other components. In other words, each group ofnanoparticles may have a different targeting molecule valency. Thegroups may be tested in vitro to determine an optimal valency or numberof targeting molecules per nanoparticle. Nanoparticles with the optimaltargeting molecule valency may then be prepared for administration to apatient in need thereof. Optionally, nanoparticles may be tested invitro by methods known in the art and/or as described above to confirmcell uptake of the nanoparticles.

At block 1411, the nanoparticles may be administered to a patient inneed thereof (e.g., a patient having a cell/tissue of the kind targetedfor treatment). In some embodiments, the nanoparticles may beadministered by intravenous injection. Optionally, the nanoparticles maybe administered in a dose that is determined based at least on arecommended or standard dose of the therapeutic agent alone. Forexample, the nanoparticles may be administered at a dose that isequivalent to (i.e., contains as many molecules of the therapeutic agentas) 10-20%, 20-30%, 30-40%, 40-50%, 50-75%, or 75-100% of therecommended or standard dose of the therapeutic agent alone.Alternatively, the nanoparticles may be administered in a dose that isdetermined based at least in part on toxicity data. As described above,delivery of a therapeutic agent via nanoparticles may result in reducedtoxicity as compared to administration of the therapeutic agent alone.Thus, in some examples, the nanoparticles may be administered in a dosethat is equivalent to 100-200% or more of the recommended or standarddose of the therapeutic agent alone. In some examples, the nanoparticlesmay be administered in a dose that is equivalent to 150% or more of therecommended or standard dose of the therapeutic agent alone.

At block 1413, the effect of the nanoparticles on the target cell ortissue may be measured. In some embodiments, the effect of thenanoparticles may be measured by measuring the size of a tumor beforeadministration of the nanoparticles, measuring the size of the tumor atone or more time points after the administration of the nanoparticles,and comparing the measurements. In other embodiments, the effect of thenanoparticles may be measured by other known methods.

Thus, embodiments of the present disclosure provide nanoparticles havinga hydrophobic interior portion surrounded by an outer portion, atherapeutic agent coupled to, and disposed at least partially within,the outer portion, and a targeting agent coupled to, and disposed atleast partially within, the outer portion. The outer portion maycomprise a water-soluble polymer. The nanoparticle may be a micellarnanoparticle. The water-soluble polymer may be coupled to a first lipid,the therapeutic agent may be coupled to a second lipid, and thetargeting agent may be coupled to a third lipid. The first, second, andthird lipids may be disposed within the interior portion of thenanoparticle. The water-soluble polymer may be polyethylene glycol(PEG). The therapeutic agent may be an antibiotic or an anti-cancerdrug, such as doxorubicin. The targeting agent may comprise a peptide(e.g., VLA-4-pep; SEQ ID NO: 1). The targeting agent may be configuredto bind to a receptor of a target cell. The nanoparticle may have a sizeof about 20 nm, and may include 10 to 20 or 20 to 40 molecules of thetargeting agent. In some examples, the water-soluble polymer comprisespolyethylene glycol (PEG), and the therapeutic agent is coupled to thesecond lipid via a pH-sensitive bond. In a specific example, thetargeting agent is configured to bind to a VLA-4 receptor of a targetcell, the therapeutic agent comprises doxorubicin, and the pH-labilebond is configured to hydrolyze at the pH of an endocytic vesicle of thetarget cell.

Other embodiments of the present disclosure provide methods ofdelivering a therapeutic agent to a cell or tissue of interest in anindividual. One method may comprise administering to the individual ananoparticle having a hydrophobic interior portion surrounded by anouter portion, a therapeutic agent coupled to, and disposed at leastpartially within, the outer portion; and a targeting agent coupled to,and disposed at least partially within, the outer portion. The targetingagent of the nanoparticle may enhance accumulation of said therapeuticagent in the cell or tissue of interest. The targeting agent may bind toa receptor of the cell or tissue of interest, and the targeting agentmay enhance receptor-mediated endocytosis of the nanoparticle by thecell or tissue of interest. The cell or tissue of interest may be acancerous cell or tissue, such as a multiple myeloma cell. In someexamples, the targeting agent is VLA-4-pep (SEQ ID NO: 1) and thetherapeutic agent is doxorubicin.

Other embodiments of the present disclosure provide a pharmaceuticalcomposition comprising a nanoparticle and a pharmacologically acceptableexcipient. The nanoparticle may have a hydrophobic interior portionsurrounded by an outer portion, a therapeutic agent coupled to, anddisposed at least partially within, the outer portion, and a targetingagent coupled to, and disposed at least partially within, the outerportion.

Other embodiments of the present disclosure provide nanoparticles forthe preparation of a pharmaceutical composition for the treatment of acancer. The nanoparticles may comprise a hydrophobic interior portionsurrounded by an outer portion, a therapeutic agent coupled to, anddisposed at least partially within, the outer portion, and a targetingagent coupled to, and disposed at least partially within, the outerportion.

Other embodiments of the present disclosure provide methods ofconstructing a nanoparticle-based drug delivery system. One method maycomprise adding quantities of a first component, a second component, anda third component to a solvent in a predetermined molar ratio, whereinthe first component comprises a targeting molecule coupled to a firstlipid molecule, the second component comprises a therapeutic agentcoupled to a second lipid molecule, and the third component comprisespolyethylene glycol (PEG), and mixing said components in the solvent toform a plurality of nanoparticles that include said components in thepredetermined molar ratio. The nanoparticles may have a hydrophobicinterior portion surrounded by an outer portion. The first and secondlipid molecules may be disposed in the interior portion, and the PEG,the targeting molecule, and the therapeutic agent may be disposed in theouter portion. The method may further include coupling the targetingmolecule to the first lipid molecule to form the first component priorto said mixing. The method may also include coupling the therapeuticagent to the second lipid molecule to form the second component prior tosaid mixing. In some embodiments, the method may further includesynthesizing the targeting molecule on a solid support and cleaving thetargeting molecule from the solid support after coupling the targetingmolecule to the first lipid molecule.

The nanoparticles may be micelles with a diameter of about 20 nm. Thetargeting molecule may comprise a peptide, and coupling the targetingmolecule to the first lipid molecule may include conjugating the peptideto the lipid molecule. The therapeutic agent may be coupled to thesecond lipid molecule by a pH-sensitive bond. In some embodiments, thetherapeutic agent comprises an antineoplastic drug. The targetingmolecule may be configured to bind to a receptor of a target cell. Insome examples, the targeting molecule is VLA-4-pep (SEQ ID NO: 1) andthe therapeutic agent is doxorubicin. The third component may include athird lipid molecule coupled to the PEG. Optionally, the first lipidmolecule and/or the third lipid molecule may comprise DSPE. Thepredetermined molecular ratio of the first component to the second andthird components in the nanoparticles may be about 20:70.

Other embodiments of the present disclosure provide methods fordesigning a drug delivery system to treat a disease or other medicalcondition. One method may comprise selecting a target cell or tissue fortreatment with a first therapeutic agent, wherein the surface of theselected target cell or tissue includes a first surface receptor,selecting a ligand that binds to the first surface receptor, and addingquantities of a first component, a second component, and a thirdcomponent to a first solvent in a predetermined molar ratio to formnanoparticles. The first component may include the ligand coupled to afirst lipid. The second component may include the therapeutic agentcoupled to a second lipid. The third component may comprise polyethyleneglycol (PEG). In some embodiments, the method may further comprisecoupling the ligand to the first lipid to form the first component. Inother embodiments, the method may further comprise coupling the firsttherapeutic agent to the second lipid to form the second component. Thenanoparticle may be a micelle with a lipid core surrounded by an outerlayer. The targeting agent, the therapeutic agent, and the PEG may bedisposed in the outer layer. Optionally, the method may further comprisemeasuring an effect of the nanoparticles on the target cell or tissue.

Other embodiments of the present disclosure provide methods of treatinga disease or other medical condition. One method may includeadministering to a patient in need thereof a nanoparticle having ahydrophobic interior portion surrounded by an outer portion, atherapeutic agent coupled to, and disposed at least partially within,the outer portion; and a targeting agent coupled to, and disposed atleast partially within, the outer portion. The disease may be a cancerand the therapeutic agent may be a chemotherapeutic agent. The targetingagent may be a ligand for a cell surface receptor expressed by acancerous cell or tissue. In a particular embodiment, the disease ismultiple myeloma, the therapeutic agent is doxorubicin, and thetargeting agent is VLA-4-pep (SEQ ID NO: 1). The method may furthercomprise measuring an effect of the nanoparticles on the target cell ortissue.

Although certain embodiments have been illustrated and described herein,it will be appreciated by those of ordinary skill in the art that a widevariety of alternate and/or equivalent embodiments or implementationscalculated to achieve the same purposes may be substituted for theembodiments shown and described without departing from the scope. Thosewith skill in the art will readily appreciate that embodiments may beimplemented in a very wide variety of ways. This application is intendedto cover any adaptations or variations of the embodiments discussedherein. Therefore, it is manifestly intended that embodiments be limitedonly by the claims and the equivalents thereof.

1. A nanoparticle comprising: a hydrophobic interior portion surroundedby an outer portion, the outer portion comprising a water-solublepolymer; a therapeutic agent coupled to, and disposed at least partiallywithin, the outer portion; and a targeting agent coupled to, anddisposed at least partially within, the outer portion wherein thetargeting agent is a low-affinity, high-specificity peptide ligand of aconjugate of Formula I:

wherein 140 is a targeting agent configured to bind to a receptor of atarget cell; 112 comprises a polyethylene glycol moiety; and 124comprises a lipid having a polar moiety (110) and a non-polar moiety(108); or a salt thereof.
 2. The nanoparticle of claim 1, wherein thenanoparticle is a micellar nanoparticle.
 3. The nanoparticle of claim 1,wherein the water-soluble polymer is coupled to a first lipid, thetherapeutic agent is coupled to a second lipid, and the targeting agentis coupled to a third lipid, and the first, second, and third lipids aredisposed within the interior portion of the nanoparticle.
 4. Thenanoparticle of claim 3, wherein the water-soluble polymer ispolyethylene glycol (PEG).
 5. The nanoparticle of claim 1, wherein thetherapeutic agent comprises an antibiotic, an anti-cancer agent, or acombination thereof.
 6. The nanoparticle of claim 1, wherein thetherapeutic agent comprises a nucleic acid, doxorubicin, bortezomib,carfilzomib, cisplatin, carboplatin, or a combination thereof. 7.(canceled)
 8. (canceled)
 9. The nanoparticle of claim 1, wherein thetargeting agent comprises VLA₄-pep (SEQ ID NO: 1), CFLDFP (SEQ ID NO:2), (X)CDPC (SEQ ID NO: 3), XC(Z)PC (SEQ ID NO: 4), XCA(Z)C (SEQ ID NO:5), (X)CSPC (SEQ ID NO: 6), YC(X)C (SEQ ID NO: 7), or RC(X)PC (SEQ IDNO: 8) where X and Z are variable amino acids.
 10. The nanoparticle ofclaim 1, wherein the nanoparticle has a size of about 20 nm and includesabout 10 to about 20, about 20 to about 40, or about 80 to about 100molecules of said targeting agent.
 11. The nanoparticle of claim 3,wherein the water-soluble polymer comprises polyethylene glycol (PEG),and the therapeutic agent is coupled to the second lipid via apH-sensitive bond.
 12. The nanoparticle of claim 11, wherein thetargeting agent is configured to bind to a VLA-4 receptor of a targetcell, the therapeutic agent comprises doxorubicin, and the pH-sensitivebond is configured to hydrolyze at the pH of an endocytic vesicle of thetarget cell.
 13. A method of delivering a therapeutic agent to a cell ortissue of interest in an individual, comprising administering thenanoparticle of claim 1 to a patient in need thereof, wherein thetargeting agent of said nanoparticle enhances accumulation of saidtherapeutic agent in the cell or tissue of interest.
 14. The method ofclaim 13, wherein the targeting agent binds to a receptor of the cell ortissue of interest, and the targeting agent enhances receptor-mediatedendocytosis of the nanoparticle by the cell or tissue of interest. 15.The method of claim 13, wherein the cell or tissue of interest is acancerous cell or tissue, or bacterially infected tissue.
 16. The methodof claim 15, wherein the cell of interest is a multiple myeloma cell,leukemia cell, or lymphoma cell.
 17. The method of claim 15, wherein thetargeting agent comprises VLA₄-pep (SEQ ID NO: 1), CFLDFP (SEQ ID NO:2), (X)CDPC (SEQ ID NO: 3), XC(Z)PC (SEQ ID NO: 4), XCA(Z)C (SEQ ID NO:5), (X)CSPC (SEQ ID NO: 6), YC(X)C (SEQ ID NO: 7), or RC(X)PC (SEQ IDNO: 8) where X and Z are variable amino acids, and the therapeutic agentcomprises a nucleic acid, doxorubicin, bortezomib, carfilzomib,cisplatin, carboplatin, or a combination thereof.
 18. A pharmaceuticalcomposition comprising the nanoparticle of claim 1 and apharmacologically acceptable excipient.
 19. The method of claim 13 forthe treatment of a cancer, or a bacterial infection.
 20. A method ofconstructing a nanoparticle-based drug delivery system, the methodcomprising: adding quantities of a first component, a second component,and a third component to a solvent in a predetermined molar ratio,wherein the first component comprises a targeting molecule coupled to afirst lipid molecule, the second component comprises a therapeutic agentcoupled to a second lipid molecule, and the third component comprisespolyethylene glycol (PEG); and mixing said components in the solvent toform a plurality of nanoparticles that include said components in thepredetermined molar ratio, wherein the nanoparticles have a hydrophobicinterior portion surrounded by an outer portion, the first and secondlipid molecules are disposed in the interior portion, and the PEG, thetargeting molecule, and the therapeutic agent are disposed in the outerportion.
 21. The nanoparticle of claim 1, wherein the conjugate ofFormula I is Formula II:

wherein Tx is targeting agent.
 22. The nanoparticle of claim 1, whereinthe therapeutic agent comprises a pH-sensitive bond configured tohydrolyze at the pH of an endocytic vesicle of the target cell, andoptionally the therapeutic agent is a conjugate of Formula III.

wherein Ty is therapeutic agent; or a salt, or combination thereof.